Radioimmunoassay and other radioligand assays involve a final separation step in which bound and free radioligand are separated into different phases to permit measurement of the radioactivity associated with one of the phases. In most assays the separation involves two procedures; first, a centrifugation, and then second, a removal of the soluble phase by decantation, suction or the like. Such procedures are work intensive and they are obstacles to the development of completely automated radioimmunoassay systems.
The removal of the supernatant leads to major difficulties of inprecision in the assay and a risk of contaminating the outside of the assay test tubes. An alternative approach which would obviate these difficulties is to separate the bound from free radioligand into different phases and then selectively shield the radiation from one of the phases while both phases are still within the assay test tube. The low energy gamma-radiation of .sup.125 I (27--35 KeV), the dominating tracer of radioimmunoassays penetrates most high density materials quite poorly. By including an attenuating material in the assay, it is possible to prevent the radiation of one of the resulting phases from emerging from the tube.
U.S. Pat. No. 4,158,135 describes a method of analysis in which a radioactive substance is distributed in a liquid phase and a solid particulate phase, and radiation from one phase is measured while attenuating the radiation from the other phase by providing to the latter phase a radiation-absorbing material. In this manner the radioassay can be carried out without physically separating the two phases. The selection of suitable radiation absorbing materials depends in large part on the identity of the radioactive isotope used in the assay. Thus, for example, when the widely employed isotope .sup.125 I is utilized, an effective attenuation of radiation is achieved with a wide variety of elements including those with a relatively low Z number. Suitable elements include silver, cadium, tungsten and bismuth. The specific embodiments described in the aforementioned patent use tungsten powder as the attenuating material.
In the selection of the proper attenuating material and its physical form, it is desired that such material does not interfere in the antigen-antibody binding or, if insoluble, does not adsorb the reactants of the assay onto its surface. Insolubilization of the bound or unbound radioligand to achieve separation of these components for measurements is achieved in the aforementioned patent by utilizing techniques and reagents known in the art, i.e., double antibody separation, chemical precipitation, or by utilizing antibodies or antigens bound to water insoluble polymers in particle form. Alternatively, the unreacted radioactive material is rendered insoluble by adsorbing it to a particulate adsorber, usually active charcoal which preferably is treated with dextran. The charcoal can be physically mixed with a radiation absorbing material to effect a further embodiment in the aforementioned patent. Finally, another alternative material utilized therein is a radioactivity absorber which also absorbs the unreacted radioactive material. An example of such material is bismuth carbonate powder.